Department for Proteomics and Signal Transduction at Max-Planck Institute for Biochemistry, 82152 Martinsried, Germany.
J Proteome Res. 2010 Jul 2;9(7):3688-700. doi: 10.1021/pr100234w.
Tissue samples in biobanks are typically formalin-fixed and paraffin-embedded (FFPE), in which form they are preserved for decades. It has only recently been shown that proteins in FFPE tissues can be identified by mass spectrometry-based proteomics but analysis of post-translational modifications is thought to be difficult or impossible. The filter aided sample preparation (FASP) method can analyze proteomic samples solubilized in high concentrations of SDS and we use this feature to develop a simple protocol for FFPE analysis. Combination with simple pipet-tip based peptide fractionation identified about 5000 mouse liver proteins in 24 h measurement time-the same as in fresh tissue. Results from the FFPE-FASP procedure do not indicate any discernible changes due to storage time, hematoxylin staining or laser capture microdissection. We compared fresh against FFPE tissue using the SILAC mouse and found no significant qualitative or quantitative differences between these samples either at the protein or the peptide level. Application of our FFPE-FASP protocol to phosphorylation and N-glycosylation pinpointed nearly 5000 phosphosites and 1500 N-glycosylation sites. Analysis of FFPE tissue of the SILAC mouse revealed that these post-translational modifications were quantitatively preserved. Thus, FFPE biobank material can be analyzed by quantitative proteomics at the level of proteins and post-translational modifications.
生物银行中的组织样本通常经过福尔马林固定和石蜡包埋（FFPE），以这种形式可以保存数十年。最近才表明，基于质谱的蛋白质组学可以鉴定 FFPE 组织中的蛋白质，但据认为翻译后修饰的分析很困难或不可能。过滤辅助样品制备（FASP）方法可以分析在 SDS 高浓度下溶解的蛋白质组学样品，我们利用这一特性开发了一种用于 FFPE 分析的简单方案。结合简单的基于移液管尖端的肽分级分离，在 24 小时测量时间内鉴定了约 5000 种小鼠肝蛋白-与新鲜组织相同。FFPE-FASP 程序的结果表明，由于储存时间、苏木精染色或激光捕获显微切割，没有任何可辨别的变化。我们使用 SILAC 小鼠将新鲜组织与 FFPE 组织进行了比较，在蛋白质或肽水平上，这些样品之间没有明显的定性或定量差异。我们将 FFPE-FASP 方案应用于磷酸化和 N-糖基化，确定了近 5000 个磷酸化位点和 1500 个 N-糖基化位点。对 SILAC 小鼠的 FFPE 组织分析表明，这些翻译后修饰在定量上得到了保存。因此，FFPE 生物银行材料可以在蛋白质和翻译后修饰水平上进行定量蛋白质组学分析。
